There is limited knowledge about the relationship between the GABAA receptor subunit composition and the synaptic, extrasynaptic and other subcellular localization of this receptor. This is in spite of the importance of the GABAA receptor in brain function. It has been estimated that 40% of the brain synapses are GABAergic. The long-term objective of this proposal is to understand the molecular mechanisms involved in clustering and anchoring GABAA receptors at the postsynaptic membrane. The aims of this proposal are: 1) To identify the amino acids of various subunits involved in the synaptic clustering of the GABAA receptors; 2) To study the synaptic and extrasynaptic localization of the gamma 3 and alpha 5 subunits and 3) To biochemically separate and characterize synaptic and non-synaptic GABAA receptors from various subcellular fractions isolated from the brain. Already available subunit-specific GABAA receptor antibodies in combination with immunocytochemistry at the light microscopy and electron microscopy levels will be used for revealing the cellular, subcellular and synaptic localization of various GABAA receptors in the rat brain and in hippocampal cultures. The same antibodies will be used for the biochemical characterization of the GABAA receptors in the subcellular fractions isolated from the brain. Techniques such as siRNA treatment of cultured hippocampal neurons and the expression of mutated GABAA receptor subunits in these cultures and in the brain will also be used aiming to elucidate the mechanisms involved in the synaptic clustering of GABAA receptors. These studies are relevant for understanding the mechanisms involved in the formation, maintenance and plasticity of the inhibitory GABAergic synapses in the brain. It is predicted that the inappropriate synaptic and extrasynaptic localization of GABAA receptors affects GABAergic synaptic function and brain development, leading to neurological and mental disorders. These studies will also be relevant for designing GABAergic therapeutic drugs with higher clinical specificity and fewer side effects.